text
string | predicted_class
string | confidence
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|---|---|---|
Tau carrying the FTDP-17-associated P301S mutation has clearly different properties. In contrast to WT tau, the total level of the P301S mutant protein increases over time, indicating an effect of the mutation on tau turnover, possibly via accumulation of insoluble forms of the protein with a longer half-life42. Indeed, P301S tau displays enhanced propensity to aggregate, as demonstrated by the constant accumulation of MC1-immunoreactive tau species, and the formation of tau multimers. However, P301S tau appears to be proportionally less phosphorylated than WT tau. The only exception is the increased immunoreactivity for AT100, a phosphorylated tau epitope present at late stages of aggregation characterized by the presence of PHF. Remarkably, and in contrast to WT tau, the behavioral deficits induced by overexpression of P301S tau clearly progress as a function of age. This effect correlates with the increase in the amount of misfolded aggregated tau. The pathogenic effects of P301S tau are also underlined by the axonal damage observed in vivo (Fig. 4c), and by the neuritic swellings induced in cortical neurons exposed to vinblastine (Fig. 7d).
|
study
| 100.0 |
Overall, these results show that motifs in the microtubule-binding domain of the tau protein control the pathogenic changes caused by overexpression of human tau in vivo. By facilitating the transition towards a β-sheet conformation, the P301S mutation associated with FTDP-17 dramatically enhances tau misfolding and aggregation, despite low levels of phosphorylation, and leads to a rapidly progressing pathology. In contrast, WT 4R0N tau is more extensively hyperphosphorylated. However, the protein does not accumulate into highly aggregated forms, and the behavioral effects of the induced pathology do not progress over time. Nevertheless, it is possible to prevent these behavioral defects by inserting β-sheet-breaking proline residues in the microtubule-binding domain. This modification of the tau protein enhances interaction with microtubules and prevents hyperphosphorylation on the residues that are abundantly phosphorylated in WT tau. This region of the tau protein has therefore a critical role both in the phosphorylation and aggregation of the tau protein, and might be centrally implicated in the pathogenic mechanisms that confer toxic properties to the tau protein.
|
study
| 100.0 |
All shuttle AAV plasmids encoding each of the human tau variants were derived from the pAAV-PGK-MCS-WPRE backbone (modified from pAAV-CMV-MCS provided by Stratagene) following standard cloning procedures. The cDNA fragments encoding human 4R0N tau and the P301S mutant were kindly provided by Dr M.G. Spillantini (Centre for Brain Repair, Cambridge University). The human 2P mutant tau (2P tau) was generated by introducing missense mutations encoding the I277P and I308P amino acid substitutions in the 4R0N tau cDNA, using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies). The control vector is derived from the same backbone and encodes the maxFP fluorescent protein.
|
study
| 100.0 |
Vector production and titration were performed as previously described43. Briefly, each pAAV plasmid was co-transfected with the pDP6 helper plasmid in HEK293-AAV cells (Agilent Technologies). Transfected cells were lysed 48 h later by freeze-thaw cycles. Recovered viral particles were sequentially purified on iodixanol density gradients and heparin affinity columns. Infectivity titers were determined according to the amount of TU measured by real-time PCR, at 48 h after infection of HEK293T cells. The titers obtained for each AAV2/6 vector suspension were: 1.88E + 10 TU/ml, 1.87E + 10 TU/ml and 6.75E + 10 TU/ml for WT 4R0N tau (3 batches); 5.79E + 10 TU/ml and 5.24E + 10 TU/ml for P301S mutant 4R0N tau (2 batches); 2.43E + 10 TU/ml for 2P mutant 4R0N tau.
|
study
| 100.0 |
Mice were housed in a room with controlled temperature and maintained in a 12:12 h light:dark cycle, with ad libitum access to water and food. All experiments were performed in accordance with Swiss legislation and the European Community Council directive (86/609/EEC) for the care and use of laboratory animals and were approved by the Veterinarian Office of the canton of Vaud and a local ethics committee. Timed pregnant C57BL6/J mice were ordered from Charles River Laboratories (France).
|
other
| 99.9 |
Newborn mouse pups (C57BL6/J, males and females) were isolated from the home cage at postnatal day 3, kept on a heating pad and anesthetized with intraperitoneal injection of a cocktail containing 0.67 mg/ml medetomidine (Dorbene; Dr. Graeub AG) and 1.7 mg/ml midazolam (Dormicum; Roche) in a total volume of 3.7 μl. The pups were placed on a plastic form mounted on a stereotactic frame and which was especially designed to maintain their head in a fixed position. Simultaneous bilateral intracerebroventricular (ICV) injections were performed using 30 G sharp needles connected to a KDS310 nanopump (KD Scientific). A total dose of 1E + 7 TU of AAV2/6 vector suspension, in a maximal volume of 1.5 µl, was injected in each lateral ventricle at a speed of 1.5 μl/min. Mice receiving virus were randomized during injection day in order to have all groups represented in a given litter. Animals were returned to their home cage after anesthesia reversal with a subcutaneous injection of 5 μl of 1 mg/ml atipamezol (Alzane; Dr. Graeub AG).
|
study
| 99.94 |
For behavioral analysis, experimental groups were composed of 12 to 15 sex-matched. General motor behavior was assessed using an open field task. A white square box divided in four compartments (50 × 50 × 37 cm) was used to monitor the spontaneous activity of four mice simultaneously. Under dim and dispersed light conditions, each mouse was placed in the center of the arena and allowed to move freely for 10 min. Various parameters including the total distance moved were analyzed using an Ethovision XT (Noldus) tracking system.
|
study
| 100.0 |
Motor performance was assessed in the rotarod test using a fixed speed protocol. On day 1, mice had two 60-sec training sessions at low speed (10–20 rpm) on the rotarod (Ugo Basile). On day 2, all mice were tested in random order, with two 60-sec trials at various increasing speeds (20, 25, 30 and 35 rpm). The time until the mouse fall down from the rotating rod was measured until the end of the trial (maximal time is 60 sec). The final score for each mouse was the average of two measurements at a speed of 35 rpm. For all motor behavior tests the investigator was blinded to the group allocation of each mouse.
|
study
| 100.0 |
Animals were sacrificed with an overdose of pentobarbital and perfused transcardially with heparinized PBS. After opening the skull, the brains were carefully dissected on ice. One hemisphere was kept frozen at −80 °C for later biochemical analysis. The other hemisphere was fixed in 4% paraformaldehyde for 4 h and transferred to 25% sucrose for histological processing. 25 μm-thick sagittal cryosections were collected on Superfrost plus slides (Thermo Fisher Scientific) and stored at −80 °C until further use. For the thioflavin S staining, frozen brain sections were incubated in a solution of 0.01% thioflavin S (Sigma Aldrich) in distilled water for 8 min at RT, washed twice in 50% ethanol for 5 min, and twice in PBS for 10 min. For Gallyas staining, frozen brain sections were treated following a modified Gallyas-silver method. Slides were mounted in 80% glycerol and images of brain sections were taken on a Leica DM5500 microscope (Leica).
|
study
| 99.9 |
For the detection of various forms of tau, the following primary antibodies were used: HT7 human specific anti-tau (Thermo Fisher Scientific, MN1000), anti-phosphorylated tau AT8 (pSer202 and pThr205, Thermo Fisher Scientific, MN1020) and PHF1 (pSer396 and pSer404, kindly provided by Dr Peter Davies), anti-PHF tau MC1 (kindly provided by Dr Peter Davies) and AT100 (Thermo Fisher Scientific, MN1060).
|
other
| 93.6 |
After rehydrating the sections in PBS for 15 min at RT, endogenous peroxidases were quenched in 1/1000 phenylhydrazine (Sigma Aldrich) in PBS at 37 °C for 1 h. Sections were then washed 3 times in PBS for 10 min. The blocking of non-specific binding sites was performed by incubating the slides in 5% normal goat serum (Jackson Immunoresearch), 3% bovine serum albumin (Sigma Aldrich) and 0.1% Triton X-100 (Sigma Aldrich) in PBS for 2 h at RT. Primary antibodies were diluted at 1/1000 in blocking buffer and applied overnight at 4 °C. After 3 washes in PBS, a biotinylated goat anti-mouse (Vector Laboratories) secondary antibody diluted 1/200 in PBS was applied for 3 h at RT. The avidin/biotin Vectastain ABC Elite kit (Vector Laboratories) was used for peroxidase detection by Metal Enhanced DAB Substrate Kit (Thermo Fisher Scientific), according to manufacturer’s instructions. Slides were then washed in PBS and mounted in Eukitt mounting medium (Sigma Aldrich). Images of brain sections were taken on a Leica DM5500 microscope (Leica).
|
other
| 99.9 |
Brains were homogenized on ice using a glass potter in a 9-fold volume of homogenization buffer: 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA containing phosphatase inhibitors (30 mM NaF, 0.2 mM Na3VO4, 1 nM Okadaic acid, 1 mM PMSF, 5 mM Na4P2O7) and a cocktail of protease inhibitors (Roche). Protein concentration was measured using Micro BCA Protein assay kit (Thermo Fisher Scientific). Protein samples were aliquoted and stored at −80 °C.
|
study
| 99.94 |
For sarcosyl extraction 50 µl of brain homogenates were mixed with 85 µl of homogenization buffer (135 µl final volume). 15 µl of 100% sucrose were added to each sample to then centrifuge at 20,000 g for 20 min at 4 °C. The supernatant was collected and sarcosyl 20% was added to each sample to reach 1% final concentration. Samples were incubated under agitation for 1 h at RT and centrifuged at 100,000 g for 1 h at 4 °C. The collected supernatant corresponded to the sarcosyl soluble fraction. The pellet obtained, called sarcosyl insoluble fraction, was resuspended in 35 µl of 50 mM Tris-Cl pH 7.4.
|
study
| 99.94 |
Ten μg of proteins were loaded in each lane of a 10% Bis-Tris precast gel (NuPAGE, Life technologies) and run in NuPAGE 1x MOPS SDS running buffer (Life Technologies). After migration of the samples, the proteins were transferred on a PVDF membrane (0.45 µm) (Millipore). Membranes were blocked for 1 h in Odyssey blocking buffer, incubated overnight with primary antibodies (mouse TAU-13 or rabbit anti-actin (Abcam), washed three times with PBS 0.1% Tween-20, incubated 1 h at RT with secondary anti-Mouse-IRDye CW-800 or anti-Rabbit-IRDye CW-680 antibodies (Odyssey). The membranes were finally scanned using LICOR (Odyssey Infrared Imager) and quantification performed using the LICOR software.
|
study
| 99.6 |
For the quantification of various tau species in brain homogenates, different AlphaLisa assays were developed according to manufacturer instructions (Perkin Elmer). Acceptor beads were conjugated to either the HT7 anti-human tau antibody (Thermo Fisher Scientific, MN1000) or the TAU-13 anti-human tau (Abcam, B11E8). Detection antibodies pSer202/pThr205 (AT8, Thermo Fisher Scientific, MN1020), pThr181 (AT270, Thermo Fisher Scientific, MN1050), pThr231 (AT180, Thermo Fisher Scientific, MN1040), human tau (HT7, Thermo Fisher Scientific, MN1000), pThr212 (Santa Cruz Biotechnology), MC1 (kindly provided by Prof. Peter Davies), pSer409 and pSer396 (AC Immune) were either purchased with biotin tag or conjugated to biotin using EZ-Link™ Sulfo-NHS-Biotin (Thermo Fischer Scientific). Brain total homogenates were pre-diluted in PBS to obtain a 5 µg/µl stock concentration. Dilutions of all reagents were made with Alpha Assay buffer (PerkinElmer). Reagents were added in a 384-well white OptiPlate (PerkinElmer) to a final volume of 50 µl. 5 µl of total brain homogenate (final concentration in an assay: 0.5 µg/µl), 10 µl of biotinylated detection antibody (final concentration: 10 nM), 10 µL of tau acceptor beads conjugate (final bead concentration: 2.5 µg/ml) were incubated 1 h at RT, before the addition of 25 µl of donor beads (final bead concentration: 25 µg/ml) and a further incubation at RT for 30 min (protected from light). The plate reading was performed using EnSpire Alpha instrument and analysis using EnSpire Workstation version 3.00.
|
study
| 100.0 |
Anaesthetized animals were perfused with a buffered solution of glutaraldehyde (2.5%) and paraformaldehyde (2%), and then vibratome sectioned at 80 µm thickness cut through the mouse cortex, close to the site of injection. Selected sections were then further fixed with 1.5% potassium ferrocyanide and 1% osmium tetroxide, followed by 1% osmium alone, and then uranyl acetate. After dehydration in a series of ascending concentrations of ethanol, they were embedded in durcupan resin and hardened at 65 °C for 24 h. Thin sections at 50 nm thickness were cut through the region of interest, stained with lead citrate and uranyl acetate, and imaged in a transmission electron microscope at 80 kV (Tecnai Spirit, FEI Company). To quantify microtubule number and density, 30 axonal cross-sections per sample were randomly analyzed in the mouse cortex, using the TrakEM2 plugin (Fiji)44. For each axon, the number of microtubules was counted and divided by the measured cross-section area to determine microtubule density.
|
study
| 100.0 |
Frontal cortex tissue was dissected from E16 C57BL/6 J mouse embryos following standard methods. We performed AAV2/6 infections at DIV1 to induce transgene expression using a viral dose of 2.5E + 5 TU per well. For this experiment, we used a bicistronic vector construct co-expressing a nuclear GFP to identify the transduced neurons (AAV2/6-PGK-Tau:CMV-nlsGFP). To determine the effects of human tau variants in presence of vinblastine, AAV2/6-infected neurons were incubated at DIV4 during 24 h or 7 days with 1 nM vinblastine and fixed with 4% PFA at DIV5 or DIV11. AAV2/6-transduced mouse cortical neurons overexpressing human tau were immunostained using the HT7 (Thermo Fisher Scientific, MN1000) and GFP (Thermo Fisher Scientific, A-11122) antibodies. For morphological analysis, all conditions were performed in triplicate.
|
study
| 100.0 |
For the quantification of filopodia, we took from each coverslip five high-magnification images (40x) where individual neurites could be visualized. To ensure comparable abundance of human tau for each condition, we assessed the morphology of neurites with similar intensity of the human tau immunostaining. A total of 15 images were analyzed for each condition in a blind manner. The number of filopodia present per 100 µm of neurite length was measured using ImageJ.
|
study
| 100.0 |
To quantify neuritic swellings, five low-magnification images (20x) of individual neurites were taken from each coverslip, with similar intensity of the human tau immunostaining. A total of 15 low-magnification images were analyzed for each condition in a blind manner. For each image, the number of swellings present per 100 µm of neurite length was measured using ImageJ.
|
study
| 99.94 |
All statistical analyses were performed using the Statistica V.6 software and graphs were prepared using Microsoft Excel. Student’s t test, Kruskal-Wallis test, and two-way ANOVA followed by different post-hoc tests were used to compare different groups. Parametric Student’s or ANOVA tests were applied to normally distributed data sets with equal variance. Since this study used a new animal model, we did not have any preliminary data set to decide the sample size based on a statistical method. Hence, we have estimated the sample size based on literature and our previous experience with similar models. No animals were excluded from the study for the data analysis. For all graphs data represent mean ± SEM.
|
study
| 100.0 |
To avoid complications after percutaneous coronary intervention (PCI), such as acute and late thrombosis at the site of stenting and recurrent ischemic cardiovascular events, anti-platelet therapy is crucial . Several studies [2–4] have shown that dual anti-platelet therapy with aspirin and a thienopyridine ADP-receptor blocker is effective in preventing such acute and late complications. Clopidogrel is an ADP-receptor blocker that is used regularly in Japan. It has a better safety profile than ticlopidine, a previous-generation ADP-receptor blocker, but its main disadvantage is the wide interindividual variations of its anti-platelet effect . Hoshino et al. evaluated the anti-platelet effect of clopidogrel in Japanese patients and found wide interindividual variation as well as a proportion (approximately 14%) of clopidogrel non-responders .
|
review
| 99.75 |
Prasugrel is a next-generation thienopyridine anti-platelet agent that has been approved in over 80 countries for patients with acute coronary syndrome (ACS) undergoing PCI. Prasugrel provides more prompt, potent, and consistent platelet inhibition than clopidogrel, and the effects of prasugrel are not influenced by the presence of CYP2C19 polymorphisms . The efficacy and safety of prasugrel were confirmed in Japanese patients in two phase III studies [8, 9]. Based on these results, prasugrel was approved in Japan in March 2014 for ACS [including unstable angina, non-ST-segment elevation myocardial infarction (NSTEMI), and ST-segment elevation myocardial infarction (STEMI)], stable angina, and old myocardial infarction that requires PCI. The approved doses of prasugrel, which are exclusive for Japanese patients, are 20 mg as the initial loading dose (LD) and 3.75 mg/day as the maintenance dose (MD), which are lower than those used in Western countries (LD/MD: 60/10 mg/day). Given the higher average age and lower body weight of Japanese patients compared with Western patients, we considered that lower doses of prasugrel may effectively lower the risk of bleeding during dual anti-platelet therapy in Japanese patients while maintaining more consistent platelet inhibition than clopidogrel.
|
study
| 99.94 |
At present, data on the safety and efficacy of prasugrel in Japanese patients are limited to the populations of the phase II/III clinical trials, in which patients were selected based on strict inclusion criteria. In addition, because bleeding adverse events (AEs) have been reported as the most common adverse drug reactions (ADRs) in clinical trials, safety information should be made available in clinical settings as soon as possible by determining the incidence and severity of bleeding AEs under actual conditions of use. Therefore, this early postmarketing observational study (PRASFIT-Practice I) aimed to evaluate the safety and efficacy of the short-term use of prasugrel in patients with ACS in real-world clinical practice settings in Japan.
|
study
| 99.8 |
Briefly, this study was conducted as a postmarketing observational study in accordance with the Good Postmarketing Study Practice Guideline (Ministry of Health, Labour and Welfare Ordinance No. 171). At each institution, consecutive patients who met the inclusion criteria were enrolled prospectively. To gather as much information as possible in the period immediately after launch, retrospective data were collected for patients treated with prasugrel before the conclusion of the contract with each institution.
|
study
| 99.94 |
All 98 participating institutions approved the study protocol. Case report forms (CRFs) were collected for each patient who started treatment with prasugrel at least 1 month before the end of the study period, between 27 May 2014 and 26 January 2015. Because all patients were to be followed up until the end of the study period (26 January 2015) regardless of completion or discontinuation of prasugrel treatment, the observation period varied for each patient. Patients with ACS who were to undergo or had recently undergone PCI and had started prasugrel treatment at least 1 month before the end of the study period were included in this study.
|
study
| 99.94 |
Dosage and administration according to the Japanese prescription label of prasugrel are as follows: prasugrel should be initiated with a single 20-mg oral dose and then continued at a 3.75-mg once-daily oral dose as a maintenance dose . Prasugrel was administered as 3.75- and 5-mg tablets, in combination with aspirin (81–100 mg/day; up to 324 mg could be used as an LD). Patients receiving a prasugrel dose of 3.75 mg during approximately 5 days prior to PCI did not require an initial LD. The extent of the exposure to prasugrel and the timing of prasugrel administration (before, during, or after PCI) under the actual conditions of use were examined.
|
study
| 96.56 |
Patient demographics, clinical baseline characteristics, clinical findings assessed prior to prasugrel treatment (or before initial PCI), during hospitalization, and at discharge, and the final diagnosis of ACS were assessed. The extent of exposure to prasugrel was based on the time and date of administration of the LD, the MD, the daily dose, the duration of treatment, and continuation or discontinuation of treatment. Other variables assessed were use of other anti-platelet agents/anti-coagulants and other concomitant medications, invasive procedures other than PCI/coronary artery bypass graft, initial coronary angiography (CAG) findings if the patient underwent CAG, timing of prasugrel administration (before, during, or after PCI), vital signs, laboratory data, AEs, bleeding AEs, and cardiovascular events.
|
study
| 99.94 |
The safety outcomes assessed were the incidence of ADRs, serious ADRs, and bleeding AEs. The incidence of bleeding AEs was also assessed by clinical characteristics. AEs were defined as any unfavorable or unintended sign (including an abnormal laboratory finding), symptom, or disease showing a temporal association with the use of the study drug, irrespective of whether it was considered to be related to the drug. ADRs were defined as AEs for which a relationship to prasugrel could not be ruled out. AEs or ADRs that satisfied the following criteria were classified as serious: an event which (1) results in death, (2) is life-threatening, (3) requires hospitalization or prolongation of hospitalization, (4) results in disability or significant incapacity, (5) has the potential to result in disability or significant incapacity, (6) is as serious as any of the outcomes listed above, or (7) causes a congenital anomaly or birth defect. Detailed definitions of bleeding and cardiovascular events are provided in Electronic Supplement 1. Regarding the incidence of bleeding AEs by clinical characteristics, we identified clinical characteristics potentially affecting the incidence of bleeding AEs by comparing patients with bleeding AEs with those without.
|
study
| 99.94 |
The efficacy outcomes were the incidence of major adverse cardiovascular events (MACE). MACE was defined as a composite of cardiovascular death, non-fatal myocardial infarction (MI), and non-fatal ischemic stroke. All-cause death, non-fatal stroke, readmission due to angina pectoris, urgent revascularization, and stent thrombosis (defined as definite or probable according to the Academic Research Consortium) were also assessed.
|
other
| 94.6 |
The planned sample size was 500 patients based on the estimated number of patients who were anticipated to receive treatment with prasugrel and on enrollment feasibility. We enrolled consecutive patients in each institution to avoid patient selection bias. For each of the safety and efficacy variables, a point estimate and its 95% confidence interval (CI) were calculated.
|
study
| 99.8 |
The Chi-square test was used for subgroup analyses to identify clinical characteristics potentially affecting the incidence of bleeding AEs. The significance level was set to α = 0.05 (two-sided). All statistical analyses were performed with SAS 9.2 (SAS Institute Inc., Cary, NC, USA).
|
study
| 99.94 |
CRFs were collected from 749 patients at 98 institutions nationwide (Fig. 1). Of these, 732 patients were included in the safety analysis set, excluding those who fell under “breaches of contract” and “protocol deviations”. All 732 patients were included in the efficacy analysis set. The mean (±standard deviation) observation period was 64.9 ± 73.8 days [median (range) 31.0 (1–531) days], regardless of continuation or discontinuation of prasugrel treatment.Fig. 1Patient disposition. ACS acute coronary syndrome
|
other
| 64.9 |
Baseline demographic and clinical characteristics of patients are shown in Table 1. In the study population, 60.0% of patients had STEMI. Furthermore, 6.7% of patients had severe cardiovascular disease, classified as Killip Class IV. These patients were excluded from PRASFIT-ACS , a phase III clinical trial conducted in Japanese patients with ACS.Table 1Baseline demographic and clinical characteristics of patientsPRASFIT-Practice I N (%)(N = 732)[Reference]PRASFIT-ACS N (%)(N = 685)Sex Male560 (76.5)536 (78.2)Age (years) ≥75221 (30.2)165 (24.1) Mean ± SD67.0 ± 12.465.4 ± 11.4 Median (range)67 (29–97)65 (32–95)Body weight (kg) ≤5094 (12.8)85 (12.4) Mean ± SD63.8 ± 12.564.2 ± 12.3Final diagnosis STEMI439 (60.0)340 (49.6) NSTEMI92 (12.6)187 (27.3) Unstable angina198 (27.0)156 (22.8)Killip classification Class I572 (78.1)NA Class II91 (12.4) Class III15 (2.0) Class IV49 (6.7)Exclusion criteriaMedical history Prior MI71 (9.7)34 (5.0) Prior revascularizations92 (12.6)40 (5.8) Prior CABG7 (1.0)6 (0.9) Prior TLR33 (4.5)15 (2.2) Prior ischemic stroke37 (5.1)Exclusion criteriaComplications Hypertension559 (76.4)495 (72.3) Dyslipidemia565 (77.2)516 (75.3) Diabetes mellitus267 (36.5)250 (36.5) History of smoking249 (34.0)273 (39.9) On dialysis15 (2.0)Exclusion criteriaAntithrombotic agent Prasugrel + aspirin678 (92.6)685 (100.0) Prasugrel + aspirin + WF or DOAC19 (2.6)Exclusion criteria Prasugrel + NSAIDs (w/o aspirin)11 (1.5)Exclusion criteriaConcomitant drug PPIs347 (47.4)282 (41.2)Stent type Drug-eluting stent671 (91.7)291 (42.5)Puncture site Brachial23 (3.1)22 (3.2) Radial374 (51.1)285 (41.6) Femoral315 (43.0)366 (53.4) SD standard deviation, STEMI ST-segment elevation myocardial infarction, NSTEMI non-ST-segment elevation myocardial infarction, MI myocardial infarction, CABG coronary artery bypass graft, TLR target lesion revascularization, WF warfarin, DOAC direct oral anti-coagulant, NSAIDs non-steroidal anti-inflammatory drugs, PPIs proton pump inhibitors, NA not available
|
study
| 99.9 |
SD standard deviation, STEMI ST-segment elevation myocardial infarction, NSTEMI non-ST-segment elevation myocardial infarction, MI myocardial infarction, CABG coronary artery bypass graft, TLR target lesion revascularization, WF warfarin, DOAC direct oral anti-coagulant, NSAIDs non-steroidal anti-inflammatory drugs, PPIs proton pump inhibitors, NA not available
|
other
| 99.94 |
Regarding other clinical characteristics that were excluded from the PRASFIT-ACS , 5.1% of patients had a history of ischemic stroke; 2.0% were on dialysis; 2.6% were concomitantly using warfarin or direct oral anti-coagulants (DOACs); and 1.5% were using non-steroidal anti-inflammatory drugs (NSAIDs). The radial puncture site was the most common in the present observational study, though the femoral puncture site was the most common in PRASFIT-ACS .
|
study
| 100.0 |
Treatment status of prasugrel and discontinuations are shown in Fig. 2. An initial LD was administered to 95.1% of patients. In the majority of patients, the LD was given before the initial PCI. In 99.0% of patients, the initial prasugrel LD was 20 mg. One out of 690 (0.1%) patients was given an MD of 2.5 mg/day; the remaining patients received an MD of 3.75 mg once-daily.Fig. 2 a Timing of loading, loading dose (LD), and starting maintenance dose (MD); b duration of prasugrel treatment and reasons for discontinuation. PCI percutaneous coronary intervention, CABG coronary artery bypass grafting
|
study
| 89.75 |
Almost one-half of the patients completed or discontinued the treatment within 1 month. The most common reason for discontinuation was switching to other anti-platelet agents (70.2%, 354/504). Of 504 patients, 40 (7.9%) discontinued prasugrel treatment because of AEs.
|
other
| 96.9 |
The incidence of ADRs was 8.6% (63/732); serious ADRs, 3.4% (25/732); and bleeding AEs, 6.4% (Electronic Supplement 2). The most common ADRs were gastrointestinal disorders (e.g., gastrointestinal hemorrhage) [3.3% (24/732)]. The most common serious ADRs were also gastrointestinal disorders [2.0% (15/732)]. Table 2 summarizes the breakdown of bleeding AEs. Bleeding AEs occurred in 6.4% of patients. The most common bleeding AE was gastrointestinal disorders (2.7%), followed by general disorders and administration site conditions (1.0%). Regarding puncture site bleeding (puncture site hemorrhage or vessel puncture site hematoma), the puncture site locations were femoral (three patients), radial (two patients), femoral + radial (one patient), and brachial (one patient). The incidence of major bleeding [thrombolysis in myocardial infarction (TIMI) criteria] AEs was 1.6%. Approximately 60% (7/12) of all major bleeding AEs were gastrointestinal disorders.Table 2Incidence of bleeding adverse events by severity and siteItemResultNo. of patients in the safety analysis set732No. of patients with bleeding AEs47No. of bleeding AEs52Incidence of patients with bleeding AEs (%)6.4No. of patients with major bleeding AEs12Incidence of patients with major bleeding AEs (%)1.6Type of bleeding AENo. of patients with bleeding AEs; No. of bleeding AEsa (%)Classification of bleeding (TIMI)Major bleeding (N = 12)Minor bleeding (N = 15)Clinically relevant (N = 12)Other (N = 10)Blood and lymphatic system disorders5 (0.7)13–1 Anemia5 (0.7)13–1Eye disorders1 (0.1)–––1 Conjunctiva hemorrhage1 (0.1)–––1Cardiac disorder3 (0.4)––3– Cardiac tamponade1 (0.1)––1– Myocardial hemorrhage2 (0.3)––2–Vascular disorders2 (0.3)–1–1 Hematoma1 (0.1)–––1 Bleeding1 (0.1)–1––Respiratory, thoracic and mediastinal disorders3 (0.4)12 Epistaxis1 (0.1)––1– Hemoptysis1 (0.1)––1– Pulmonary hemorrhage1 (0.1)1–––Gastrointestinal disorders20 (2.7)7662 Hemorrhagic intestinal diverticulum1 (0.1)––1– Gastrointestinal hemorrhage6 (0.8)312– Gingival bleeding1 (0.1)–––1 Hematemesis1 (0.1)–1–– Hematochezia1 (0.1)––1– Mallory-Weiss syndrome1 (0.1)–1–– Melena4 (0.5)–121 Rectal hemorrhage2 (0.3)2––– Upper gastrointestinal hemorrhage1 (0.1)1––– Large intestinal hemorrhage1 (0.1)–1–– Duodenal hemorrhage1 (0.1)1––– Hemorrhoidal bleeding1 (0.1)–1––Skin and subcutaneous tissue disorders4 (0.5)–4–– Subcutaneous hemorrhage4 (0.5)–4––Renal and urinary disorders3 (0.4)111– Hematuria3 (0.4)111–General disorders and administration site conditions7 (1.0)22–3 Puncture site hemorrhage6 (0.8)12–3 Vessel puncture site hematoma1 (0.1)1–––Injury, poisoning and procedural complications3 (0.4)–––3 Subcutaneous hematoma2 (0.3)–––2 Wounds1 (0.1)–––1For SOC, the number of patients with bleeding AEs was tabulated, and for preferred term, the number of bleeding AEs (i.e., the number of patients for each preferred term) was tabulated. MedDRA/J version 18.1 AEs adverse events, SOC system organ class, TIMI thrombolysis in myocardial infarction aThe number of patients for SOC and the number of bleeding AEs for each preferred term were tabulated. The number of serious bleeding AEs is specified in square brackets in the applicable cells
|
study
| 91.7 |
The incidence of bleeding AEs by clinical characteristics is shown in Table 3. The incidence of bleeding AEs was significantly higher in female patients, patients aged 75 years or older, patients with low body weight (50 kg or less), patients with severe cardiovascular disease (Killip Class III or IV), patients without dyslipidemia, and patients with severe renal impairment (creatinine clearance less than 30 mL/min). The proportion of female patients weighing 50 kg or less who experienced bleeding AEs was 40.9% (9/22), compared with 16.0% (4/25) among male patients with bleeding AEs weighing 50 kg or less. Furthermore, puncture site hemorrhage and subcutaneous hemorrhage were reported more frequently in female patients [2.9% (5/172) and 1.7% (3/172), respectively], than in males [0.2% (1/560) and 0.2% (1/560)]. In contrast, variations in timing of the initial LD (before, during, or after PCI) did not significantly affect the occurrence of bleeding AEs.Table 3Incidence of bleeding adverse events by clinical characteristicPatients, N Patients with bleeding AEs, N (%) P value*Safety analysis set73247 (6.4)–Sex Male56025 (4.5)<0.0001 Female17222 (12.8)Age (years) <7551122 (4.3)0.0004 ≥7522125 (11.3)Body weight (kg)a ≤509413 (13.8)0.0008 >5061030 (4.9)Final diagnosisa STEMI43933 (7.5)0.4138 NSTEMI923 (3.3) UAP19811 (5.6)Killip classificationa Class I57229 (5.1)0.0302 Class II918 (8.8) Class III152 (13.3) Class IV497 (14.3)Prior MIa Absent65344 (6.7)0.1983 Present712 (2.8)Prior revascularizationsa Absent63443 (6.8)0.1951 Present923 (3.3)Prior CABG Absent72547 (6.5)0.4862 Present70 (0.0)Prior TLR Absent69945 (6.4)0.9312 Present332 (6.1)Prior ischemic strokea Absent68845 (6.5)0.3508 Present371 (2.7)Hypertension Absent17311 (6.4)0.9694 Present55936 (6.4)Dyslipidemia Absent16720 (12.0)0.0009 Present56527 (4.8)Diabetes mellitus Absent46528 (6.0)0.5609 Present26719 (7.1)History of smokinga Absent46236 (7.8)0.0841 Present24911 (4.4)Baseline Ccra (mL/min) Normal (>80)2999 (3.0)0.0043 Mild (>50 to ≤80)23818 (7.6) Moderate (≥30 to ≤50)979 (9.3) Severe (<30)487 (14.6)Timing of loadinga Before PCIb 53337 (6.9)0.5973 During PCIc 713 (4.2) After PCId 613 (4.9)Prasugrel + aspirin Not used543 (5.6)0.7875 Used67844 (6.5)Prasugrel + aspirin + WF or DOAC Not used71345 (6.3)0.4595 Used192 (10.5)Prasugrel + NSAIDs (w/o aspirin) Not used72147 (6.5)0.3814 Used110 (0.0)PPIs Not used38523 (6.0)0.6035 Used34724 (6.9) NSTEMI non-ST-segment elevation myocardial infarction, STEMI ST-segment elevation myocardial infarction, UAP unstable angina pectoris, MI myocardial infarction; CABG coronary artery bypass graft, TLR target lesion revascularization, Ccr creatinine clearance, WF warfarin, DOAC direct oral anti-coagulant, NSAIDs non-steroidal anti-inflammatory drugs, PPIs proton pump inhibitors, PCI percutaneous coronary intervention* χ 2 test aBody weight, Killip class, prior MI, prior revascularization, prior ischemic stroke, history of smoking, and timing of loading dose were unknown in 28, 5, 8, 6, 7, 21, and 31 patients, respectively. Three patients had a final diagnosis other than STEMI, NSTEMI, or UAP. Baseline Ccr was not calculated in 50 patients bGiven before the initial balloon passage in PCI cGiven from the initial balloon passage in PCI until discharge from the PCI room dGiven after discharge from the PCI room
|
study
| 99.94 |
NSTEMI non-ST-segment elevation myocardial infarction, STEMI ST-segment elevation myocardial infarction, UAP unstable angina pectoris, MI myocardial infarction; CABG coronary artery bypass graft, TLR target lesion revascularization, Ccr creatinine clearance, WF warfarin, DOAC direct oral anti-coagulant, NSAIDs non-steroidal anti-inflammatory drugs, PPIs proton pump inhibitors, PCI percutaneous coronary intervention
|
other
| 99.94 |
aBody weight, Killip class, prior MI, prior revascularization, prior ischemic stroke, history of smoking, and timing of loading dose were unknown in 28, 5, 8, 6, 7, 21, and 31 patients, respectively. Three patients had a final diagnosis other than STEMI, NSTEMI, or UAP. Baseline Ccr was not calculated in 50 patients
|
other
| 97.1 |
Of the above risk factors, each score for the following five main risk factors [female sex, age of 75 years or older, low body weight (50 kg or less), severe cardiovascular disease (Killip Class III or IV), and severe renal impairment (creatinine clearance less than 30 mL/min)] is defined as 1. We calculated the total risk score and the incidence of bleeding AEs. By this analysis (Fig. 3), we found that the bleeding risk increased sharply in patients who had four or all five risk factors.Fig. 3Bleeding adverse events by number of risk factors. AEs adverse events, Ccr creatinine clearance. *The score of the risk factors was not calculated in 55 patients
|
study
| 99.94 |
The details of the efficacy analysis are shown in Table 4. The incidence of MACE in the efficacy analysis was 1.9% during prasugrel treatment, and 3.1% up to the end of the observation period. Cardiovascular death was the most common MACE, occurring in 13 patients with an incidence of 1.8% at the end of the study period. Of these, eight patients had severe cardiovascular disease (Killip Class IV). The incidence of all-cause death was 1.1% during prasugrel treatment and 2.2% up to the end of the study. Cardiovascular death accounted for approximately three-fourths of all-cause death.Table 4Incidence of cardiovascular eventsEfficacy outcomesCumulative incidence (%) N = 732On treatmentUntil the end of the observation period (EAS)MACE14 (1.9)23 (3.1) CV death6 (0.8)13 (1.8) Non-fatal MI5 (0.7)5 (0.7) Non-fatal ischemic stroke3 (0.4)5 (0.7)All-cause death8 (1.1)17 (2.3)Non-fatal stroke3 (0.4)6 (0.8)Readmission due to angina pectoris4 (0.5)8 (1.1)Revascularization10 (1.4)16 (2.2)Stent thrombosis2 (0.3)5 (0.7) EAS efficacy analysis set, MACE major adverse cardiovascular events, CV cardiovascular, MI myocardial infarction
|
other
| 60.7 |
This postmarketing observational study assessed the safety and efficacy of short-term treatment with prasugrel in patients with ACS in real-world clinical practice settings in Japan. We consider that this study provides relevant information in terms of the efficacy and safety of prasugrel as we included patients with severe cardiac disease (Killip IV) (6.7% of patients), history of ischemic stroke (5.1%), and severe renal impairment (on dialysis) (2.0%), as well as those concomitantly taking drugs that increase the tendency of bleeding AEs, such as warfarin/DOACs (2.6%) or NSAIDs (1.5%). Patients having these baseline demographics and/or taking these concomitant drugs (approximately one-sixth of the patients) were excluded from clinical studies in Japan, such as PRASFIT-ACS .
|
study
| 99.94 |
In approximately 99% of patients, the initial prasugrel LD and MD were 20 and 3.75 mg, respectively; 95.1% of patients received an initial LD. Prasugrel was administered as described in the package insert , and aspirin was used concomitantly in most patients. Nearly 70% of prasugrel treatment completions or discontinuations occurred as patients switched to other anti-platelet agents. Because prasugrel was only allowed to be prescribed for a period of 2 weeks during the first year after its launch, these patients were prescribed other anti-platelet agents for subsequent long-term treatment.
|
other
| 99.9 |
The incidence of ADRs was 8.6%, and the incidence of serious ADRs was 3.4%. The highest incidence of ADRs was for gastrointestinal disorders (3.3%). Most of the ADRs and serious ADRs were bleeding AEs. A total of 12 patients experienced major bleeding AEs. Approximately 60% (seven patients) of all major bleeding AEs were gastrointestinal disorders. This finding was consistent with the observation in a survey of clopidogrel (J-PLACE) in NSTEMI/unstable angina pectoris patients scheduled to undergo PCI , which suggests that gastrointestinal disorders are the main bleeding AEs in ACS patients. Therefore, preventive measures for gastrointestinal disorders might be required. Aspirin is highly likely to be a contributor to the development of these gastrointestinal disorders and the prevention of low-dose aspirin-associated upper gastrointestinal injuries by proton pump inhibitors (PPIs) has been reported . However, in this study, the proportion of patients receiving PPIs was less than half (47.4%) of the total population assessed. Although no significant difference was noted in the incidence of bleeding AEs between patients treated with or without concomitant PPIs in this study, concomitant use of PPIs from the start of dual anti-platelet therapy seems essential for preventing gastrointestinal disorders, especially in high-risk patients.
|
study
| 99.75 |
The incidence of major bleeding AEs in patients treated with prasugrel was 1.9% in PRASFIT-ACS , and was slightly lower in this study (1.6%). Furthermore, the incidences of minor bleeding AEs, clinically relevant bleeding AEs, other bleeding AEs, and all bleeding AEs were all lower in this study in comparison with PRASFIT-ACS . One possible explanation for these differences is that, in this study, intraoperative bleeding of the expected amount associated with invasive procedures, such as PCI, was not reported as an AE. Another possible explanation is that the observation period in this study differed from that in PRASFIT-ACS .
|
study
| 99.94 |
Notably, the incidence of bleeding AEs in this study was significantly higher in female patients and patients with severe cardiovascular disease (Killip Class III or IV), in addition to patients aged 75 years or older, patients with low body weight (50 kg or less), and patients with severe renal impairment. For the elderly, patients with low body weight, and patients with severe renal impairment, prasugrel treatment should be administered with caution as specified in the “Careful Administration” section of the package insert. Women generally have lower body weight than men, and there are differences in skin tissue structure between the sexes. Therefore, the fact that a higher proportion of female patients reported subcutaneous hemorrhage is likely to be related to these observed sex differences. In fact, the second most common bleeding AE was general disorders and administration site conditions (e.g., puncture site hemorrhage) (1.0%); thus, measures to prevent puncture site bleeding might also be required. Results of the MATRIX Access study , a clinical trial conducted in European ACS patients who were about to undergo CAG and PCI, suggested that radial access compared with femoral access decreased the net AEs through a reduction in major bleeding AEs and death. Furthermore, in PRASFIT-ACS, the incidence of puncture site bleeding during PCI was lower in the radial access route group than in the femoral access route group . In this study, there was no difference in the number of patients with puncture site bleeding AEs between groups undergoing PCI via different puncture sites, which was likely because the incidence of patients with puncture site bleeding AEs was low [1.0% (7/732)], even though it was the second most common bleeding AE. The reason for the low incidence of puncture site bleeding AEs may be that—in contrast with PRASFIT-ACS—the proportion of patients undergoing PCI via femoral access was lower (43.0%) than that undergoing PCI via radial access (51.1%). Therefore, radial access seems more appropriate for preventing puncture site bleeding. In this study, the timing of the LD did not appear to significantly affect the incidence of bleeding AEs.
|
study
| 99.9 |
Though the overall incidences of bleeding AEs were lower in this study in comparison with PRASFIT-ACS , after calculating the total risk score in association with the incidence of bleeding AEs, we found that the risk of bleeding increased if patients had four or all five risk factors: “female sex”, “age of 75 years or older”, “body weight of 50 kg or less”, “severe cardiovascular disease”, and “severe renal impairment”. Another study assessed the risk of bleeding in patients with ACS undergoing PCI abroad; these investigators concluded that patients with ACS have marked variability in the risk of bleeding according to sex, age, and serum creatinine, among other factors . A study by Saito et al. , which examined periprocedural bleeding in relation to the access route for PCI in a Japanese sample, found that sex, body weight, and age were risk factors, observations that are in line with our findings. There is a possibility that the risk of bleeding will increase in the above-mentioned patients. However, an analysis adjusting for confounding effects on each risk factor was not performed. Furthermore, as a limited number of patients with severe cardiac dysfunction were evaluated in clinical trials (these patients were generally excluded), these patients will be evaluated in the ongoing PRASFIT-Practice II study, a long-term observational study in patients with ischemic heart disease.
|
study
| 99.94 |
The incidence of MACE was lower in the current study (3.1%) than in PRASFIT-ACS (9.4%). Conversely, the incidences of cardiovascular death (1.8%), all-cause death (2.2%), and non-fatal ischemic stroke (0.7%) were slightly higher in the current study than those in PRASFIT-ACS (1.3, 1.8, and 0.4%, respectively). The explanation for these differences may involve: (1) the difference in the duration of the observation period in each study, and (2) that PRASFIT-Practice I was an observational study reflecting the clinical use of prasugrel in a real-world setting. Thus, patients with severe conditions were included, whereas in the PRASFIT-ACS , such cases were excluded. The incidence of non-fatal MI was low in this study, which may also explain the low incidence of MACE compared with PRASFIT-ACS . A possible reason for this may be that naturally occurring MI as well as events judged according to CAG findings and markers of myocardial injury, including creatine kinase-MB, were evaluated in PRASFIT-ACS ; however, only cases of MI reported by investigators under the actual conditions of use were evaluated as events in this study.
|
study
| 99.94 |
This study had several limitations. Because the study was designed as a postmarketing observational study, only patients treated with prasugrel were evaluated. As this study aimed to assess the real clinical situation in Japan, patients were not subjected to strict exclusion criteria. The observation and follow-up periods varied for each patient. As this was a short-term study, the results are only applicable to patients treated during a short period. The length of the observational period was insufficient to collect an adequate number of cardiovascular events to thoroughly evaluate safety, especially in terms of risk factors. However, the long-term observational study “PRASFIT-Practice II” will address these issues.
|
study
| 99.94 |
Based on the results of this short-term clinical study in patients with ACS in a real-world acute setting, prasugrel administration at an LD of 20 mg and MD of 3.75 mg/day was considered to be acceptable for Japanese patients in terms of safety and efficacy.
|
study
| 90.75 |
Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disorder characterized by the degradation of motor neurons within the anterior horn of the spinal cord, resulting in the progressive trunk and limb muscle paralysis1. SMA is currently the most common genetic cause of infant mortality1.
|
other
| 94.4 |
Most cases of SMA are caused by homozygous loss of the survival of motor neuron 1 (SMN1) gene2. The survival of motor neuron 2 (SMN2) gene is a modifier of the SMA phenotype and has the nearly identical sequence to SMN1 with only a five-base pair difference3, 4. A C-to-T substitution in exon 7 of SMN2 leads to the skipping of exon 7 in approximately 90% of SMN2 transcripts5. The exon 7-skipped SMN2 protein cannot compensate for the SMN1 function because it is unstable and rapidly degraded. The remaining 10% of SMN2 transcripts is not sufficient to rescue the SMA phenotype. However, a higher copy number of SMN2 is associated with less severe clinical representations on average, even though it is not fully correlated1, 6.
|
study
| 99.94 |
Antisense therapy is currently one of the most promising strategies for treating SMA. Antisense oligonucleotides (AONs) are used to block intronic splicing silencer sites such as N1 (ISS-N1) on intron 7 of SMN2, which induces the inclusion of exon 7 and, consequently, leads to the recovery of functional SMN protein expression from the endogenous SMN2 gene7. A 20-mer AON with phosphorothioate backbones and 2′-O-methyl (2′OMeP) modification was the first AON to show the efficacy of AONs in vitro for SMN2 7. Nusinersen/IONIS-SMNRx (Spinraza), an 18-mer AON with phosphorothioate backbones and 2′-O-methoxyethyl modification (MOE), increased the exon 7 inclusion and rescued SMA phenotypes in vivo in mouse models8–11. Following the completion of Phase I/II trials with encouraging data, nusinersen has recently been approved by the U.S. Food and Drug Administration (FDA) and became the first drug for the treatment of SMA12–14.
|
review
| 99.9 |
Although there is much hope for this drug, it has been known to present some complications since its pre-clinical testing. For example, nusinersen is not incorporated efficiently into certain cell types and tissues8. Repeated intrathecal injection is employed for its administration in the clinical trials14, because it cannot easily cross the blood-brain barrier8. Although intrathecal injections of nusinersen are considered to be safe, the typical side effects associated with lumbar puncture are induced in nearly one-third of treated patients in the clinical trial15. In addition, the manufacturer has announced that the treatment will cost $750,000 in the first year, and $375,000 every following year16. Development of a new and more affordable alternative would bring practical benefits for the patients.
|
other
| 57.56 |
To overcome these issues, the latest research is trying to identify better antisense chemistries. Administration of phosphorodiamidate morpholino oligomers (PMOs) targeting ISS-N1 by a single intracerebroventricular injection ameliorated the SMA symptom in the mouse models17. An 8-mer 2′OMeP AONs with PEG-282 and propyl modifications at the 5′ and 3′ ends also improved the phenotypes of SMA mouse models18. Recently, it has been demonstrated that peptide-conjugated PMOs (Pip6a-PMOs) were delivered to the central nervous system (CNS) by intravenous injections and rescued the phenotype of a severe SMA mouse model19. However, no clinical trial has been reported with these chemistries for SMA.
|
review
| 99.7 |
Locked nucleic acids (LNAs) are artificial nucleic acid analogs which contain a methylene bridge connecting the 2′-O with the 4′-C position in the furanose ring (Fig. 1a)20, 21. This modification makes them resistant to nucleases and increases affinity to complementary RNA sequences. The LNA chemistry has been used for gapmer AONs, single strand DNA oligonucleotides flanked by several LNA bases at the 5′ and 3′ ends22. Gapmer AONs bind targeted mRNAs and degrade them by activation of RNase H22. LNA/DNA mixmers (AONs composed of alternating LNA and DNA nucleotides) have been recently developed, which induce exon skipping in dystrophin mRNA (LNA-based splice-switching oligonucleotides) in vitro 23, or inhibit miRNA to protect the heart against pathological cardiac remodeling to improve the heart function (LNA-antimiR) in vivo 24. Miravirsen (AntimiR-122) is an LNA-antimiR that inhibits miR-122 to treat hepatitis C infection25, 26. MRG-106 (AntimiR-155) is another LNA-AntimiR and targets miR-155 for the therapy of cutaneous T cell lymphoma and mycosis fungoides26. The clinical trials of both LNA-antimiRs are currently ongoing26. One of the advantages of using LNA/DNA mixmers for treatment is that they have negatively charged backbones. This is expected to make delivery into cells more efficient than PMOs which are neutrally charged.Figure 1LNA structure and LNA/DNA mixmer sequences. (a) Chemical structures of RNA and phosphorothioated LNA. (b) LNA/DNA mixmer sequences targeting SMN2 intron 7. DNA base: G, A, T, C. LNA base (red): +G, +A, +T, +C. Phosphorothioated DNA base: G*, A*, T*, C*. Phosphorothioated LNA base (red): +G*, +A*, +T*, +C*.
|
review
| 99.8 |
LNA structure and LNA/DNA mixmer sequences. (a) Chemical structures of RNA and phosphorothioated LNA. (b) LNA/DNA mixmer sequences targeting SMN2 intron 7. DNA base: G, A, T, C. LNA base (red): +G, +A, +T, +C. Phosphorothioated DNA base: G*, A*, T*, C*. Phosphorothioated LNA base (red): +G*, +A*, +T*, +C*.
|
other
| 99.9 |
Here, we designed a series of LNA/DNA mixmers complementary to the ISS-N1, and evaluated their ability to induce exon inclusion in type I SMA patient fibroblasts. These AONs efficiently rescued the expression of SMN protein in the cultured cells. A single mismatch in AONs significantly decreased the exon inclusion efficacy, indicating that these AONs are highly specific to the target sequence. In addition, the LNA/DNA mixmer showed significantly higher efficacy than the all-LNA oligonucleotide with the equivalent sequence. Our results demonstrate that the LNA/DNA mixmers could be promising drug candidates suitable for in vivo studies to develop AON therapies to treat SMA.
|
study
| 100.0 |
To utilize LNA/DNA mixmers for the antisense therapy for SMA, we designed eight antisense mixmers that target to the ISS-N1 in intron 7 of SMN2 (Figs 1b and 2a). AON #1, #2, #4 and #6 are LNA-based 30-mer, 18-mer, 13-mer, and 8-mer oligonucleotides, respectively. They have a DNA substitution at every other nucleotide. AON #3, #5, and #7 are 18-mer, 13-mer, and 8-mer oligonucleotides, respectively. They are also composed of LNAs, with DNA being substituted for LNA at every third nucleotide position. All nucleotides of AON #8 are LNAs. All mixmers have fully modified phosphorothioated backbones to prevent degradation by nucleases. AON #2 and #3 (18-mer) have the same sequence as nusinersen (IONIS-SMNRX) and the PMOs previously published8, 27. Shimo et al. compared the efficacies of LNA/DNA mixmers whose lengths are between 6 mer to 23 mer, and 13 mer showed the best efficacy for exon skipping23. The 8 mer was the shortest LNA/DNA mixmer which induced efficient exon skipping23. As such, we employed 13 mer and 8 mer oligos in this study.Figure 2Newly designed AONs induce SMN2 exon 7 inclusion. (a) Relative positions of newly designed AONs targeting ISS-N1 in intron 7 of SMN2. Green line: newly designed AONs (LNA/DNA mixmers). (b) The screen of AONs (LNA/DNA mixmers) by RT-PCR of SMN2 in human SMA patient fibroblasts (GM03813). Transfection of AONs was performed at 200, 100, 50, and 5 nM. Transfection of at least 5 nM of AONs #1–5 induced the exon 7 inclusion. Top band: exon 7-included. Bottom band: exon 7-excluded. M: mock control AON, NT: non-treated, H: healthy cells, B: blank.
|
study
| 100.0 |
Newly designed AONs induce SMN2 exon 7 inclusion. (a) Relative positions of newly designed AONs targeting ISS-N1 in intron 7 of SMN2. Green line: newly designed AONs (LNA/DNA mixmers). (b) The screen of AONs (LNA/DNA mixmers) by RT-PCR of SMN2 in human SMA patient fibroblasts (GM03813). Transfection of AONs was performed at 200, 100, 50, and 5 nM. Transfection of at least 5 nM of AONs #1–5 induced the exon 7 inclusion. Top band: exon 7-included. Bottom band: exon 7-excluded. M: mock control AON, NT: non-treated, H: healthy cells, B: blank.
|
study
| 100.0 |
We first determined the optimal concentration of AONs to evaluate the efficacy of exon inclusion. AONs were transfected at various concentrations to type 1 SMA patient fibroblasts (Fig. 2b). The RT-PCR analysis using SMN2 specific primers revealed that 5 nM or higher concentration of AONs #1–5 efficiently induced SMN2 exon 7 inclusion in treated cells (Fig. 2b).
|
study
| 100.0 |
Next, we examined the efficacy of each AON. AONs were transfected into the SMA patient fibroblasts at 5 nM. Semi-quantitative RT-PCR using SMN2 specific primers showed that the transfection of AONs #1–5 efficiently induced SMN2 exon 7 inclusion in the patient cells (78–98% exon 7 inclusion, Fig. 3a,b). Transfection of AONs #6–8 at this dose did not show any significant difference from the control AON. Quantitative PCR (qPCR) also showed that the levels of full-length SMN2 mRNA significantly elevated by the treatment of AONs #1–3 and #5, compared to the control (Fig. 3c).Figure 3Efficacy of the novel LNA/DNA mixmers to induce SMN2 exon 7 inclusion. 5 nM transfection of AONs #1-#5 induced SMN2 exon 7 inclusion at a significantly higher rate than the mock AON transfection. (a) RT-PCR of SMN2 and GAPDH in human SMA patient fibroblasts (GM03813). AONs #1–8 were transfected at 5 nM concentration. Top band: exon 7-included. Bottom band: exon 7-excluded. (b) Quantification of SMN2 exon 7 inclusion in the AON-treated SMA fibroblasts by RT-PCR. (c) Relative expression of full-length SMN2 to GAPDH measured by qPCR. The data were normalized to non-treated cells. Bars represent mean ± S.D. of three independent experiments. One-way ANOVA with Dunnett’s multiple comparison test. M: mock control AON, NT: non-treated, H: healthy cells, B: blank.
|
study
| 100.0 |
Efficacy of the novel LNA/DNA mixmers to induce SMN2 exon 7 inclusion. 5 nM transfection of AONs #1-#5 induced SMN2 exon 7 inclusion at a significantly higher rate than the mock AON transfection. (a) RT-PCR of SMN2 and GAPDH in human SMA patient fibroblasts (GM03813). AONs #1–8 were transfected at 5 nM concentration. Top band: exon 7-included. Bottom band: exon 7-excluded. (b) Quantification of SMN2 exon 7 inclusion in the AON-treated SMA fibroblasts by RT-PCR. (c) Relative expression of full-length SMN2 to GAPDH measured by qPCR. The data were normalized to non-treated cells. Bars represent mean ± S.D. of three independent experiments. One-way ANOVA with Dunnett’s multiple comparison test. M: mock control AON, NT: non-treated, H: healthy cells, B: blank.
|
study
| 100.0 |
In addition, Western blot protein analyses demonstrated that the levels of SMN protein significantly increased in the cells by transfection of AONs #1–5 (a 1.5–1.9-fold increase from the control, Fig. 4). The AONs #6–8 treatment did not change the levels of SMN protein in the cells. These data indicate that the LNA/DNA mixmers targeting ISS-N1 in SMN2 intron 7 induce the exon 7 inclusion efficiently in the SMA patient cells.Figure 4Transfection of the novel LNA/DNA mixmers increases the production of SMN protein. (a) Western blotting of SMN and Cofilin of healthy and AON-treated SMA patient fibroblasts (GM03813). (b) Fold increase in SMN expression of AON-treated SMA patient fibroblasts. 5 nM transfection of AON 1–5 significantly increased the production of SMN proteins. Cofilin was used as a loading control. The data was normalized to the ratio of SMN/Cofilin in non-treated SMA fibroblasts. Bars represent mean ± S.D. of four independent experiments. One-way ANOVA with Dunnett’s multiple comparison test. M: mock control AON, NT: non-treated.
|
study
| 100.0 |
Transfection of the novel LNA/DNA mixmers increases the production of SMN protein. (a) Western blotting of SMN and Cofilin of healthy and AON-treated SMA patient fibroblasts (GM03813). (b) Fold increase in SMN expression of AON-treated SMA patient fibroblasts. 5 nM transfection of AON 1–5 significantly increased the production of SMN proteins. Cofilin was used as a loading control. The data was normalized to the ratio of SMN/Cofilin in non-treated SMA fibroblasts. Bars represent mean ± S.D. of four independent experiments. One-way ANOVA with Dunnett’s multiple comparison test. M: mock control AON, NT: non-treated.
|
study
| 100.0 |
To determine the specificity of the LNA/DNA mixmers, we designed AONs containing a single mismatch in AON #5 (Fig. 5a). We compared the efficacy of AON #5 with AONs of the same sequence but including a single mismatch (AONs #10–15) by the semi-quantitative RT-PCR using SMN2 specific primers. The results indicated that all AONs containing a single mismatch showed significantly lower efficiency of exon 7 inclusion compared to AON #5 (Fig. 5b,c). Quantitative PCR (qPCR) also confirmed that the amount of full-length SMN2 mRNA did not increase by the treatment of most AONs with a single mismatch, AON #10, #11, #13, #14 and #15 (Fig. 5d). The data suggest that these LNA/DNA mixmers are highly specific to the targeted sequence, with a single mismatch leading to a significant loss of binding affinity between the mixmers and the targeted mRNAs.Figure 5A single mismatch in the LNA/DNA mixmers abrogates the activity of them for exon 7 inclusion. (a) LNA/DNA mixmer sequences used for the experiment. The LNA/DNA mixmers #10–15 contain a single base mismatch at various locations along the sequence. AON #9 consists of 100% LNA. DNA base: G, A, T, C. LNA base (red): +G, +A, +T, +C. Phosphorothioated DNA base: G*, A*, T*, C*. Phosphorothioated LNA base (red): +G*, +A*, +T*, +C*. Yellow highlighted: mismatch. (b) RT-PCR of SMN2 and GAPDH in human SMA patient fibroblasts (GM03813). AONs were transfected at 1 nM concentration. Top band: exon 7-included. Bottom band: exon 7-excluded. (c) Quantification of SMN2 exon 7 inclusion in AON-treated SMA fibroblasts by RT-PCR. ***p < 0.005. #Significant difference with the mock (#p < 0.05, ##p < 0.01, ###p < 0.005). $Significant difference with the non-treated ($p < 0.05, $$p < 0.01, $$$p < 0.005). &Significant difference with AON 4 (&p < 0.05, &&p < 0.01, &&&p < 0.005). %Significant difference with AON 11 (%p < 0.05, %%p < 0.01). (d) Relative expression of full-length SMN2 to GAPDH measured by qPCR. The data were normalized to non-treated cells. *p < 0.05, **p < 0.01, ***p < 0.005. #Significant difference with the mock (#p < 0.05, ##p < 0.01, ###p < 0.005). Bars represent mean ± S.D. of three independent experiments. One-way ANOVA with a Tukey’s multiple comparison test. M: mock control AON, NT: non-treated, H: healthy cells, B: blank.
|
study
| 100.0 |
A single mismatch in the LNA/DNA mixmers abrogates the activity of them for exon 7 inclusion. (a) LNA/DNA mixmer sequences used for the experiment. The LNA/DNA mixmers #10–15 contain a single base mismatch at various locations along the sequence. AON #9 consists of 100% LNA. DNA base: G, A, T, C. LNA base (red): +G, +A, +T, +C. Phosphorothioated DNA base: G*, A*, T*, C*. Phosphorothioated LNA base (red): +G*, +A*, +T*, +C*. Yellow highlighted: mismatch. (b) RT-PCR of SMN2 and GAPDH in human SMA patient fibroblasts (GM03813). AONs were transfected at 1 nM concentration. Top band: exon 7-included. Bottom band: exon 7-excluded. (c) Quantification of SMN2 exon 7 inclusion in AON-treated SMA fibroblasts by RT-PCR. ***p < 0.005. #Significant difference with the mock (#p < 0.05, ##p < 0.01, ###p < 0.005). $Significant difference with the non-treated ($p < 0.05, $$p < 0.01, $$$p < 0.005). &Significant difference with AON 4 (&p < 0.05, &&p < 0.01, &&&p < 0.005). %Significant difference with AON 11 (%p < 0.05, %%p < 0.01). (d) Relative expression of full-length SMN2 to GAPDH measured by qPCR. The data were normalized to non-treated cells. *p < 0.05, **p < 0.01, ***p < 0.005. #Significant difference with the mock (#p < 0.05, ##p < 0.01, ###p < 0.005). Bars represent mean ± S.D. of three independent experiments. One-way ANOVA with a Tukey’s multiple comparison test. M: mock control AON, NT: non-treated, H: healthy cells, B: blank.
|
study
| 100.0 |
Moreover, we compared the efficacies between LNA/DNA mixmers and all-LNA oligonucleotides and examined whether LNA-to-DNA substitution could improve the efficacy. When the 13 mer AONs were transfected into the cells at 1 nM, the efficiency of exon inclusion between the mixmers (AONs #4 and #5) and the all-LNA oligonucleotide with the equivalent sequence (AON #9) was not significantly different (Fig. 5b–d). However, when the 18-mer mixmers (AONs #2 and #3) and the all-LNA oligonucleotide with the equivalent sequence (AON #16) were transfected at 0.5 nM, semi-quantitative RT-PCR showed that one of the mixmers (AON #3) had a significantly higher efficacy than the all-LNA oligonucleotide (AON #16) (Fig. 6c). In addition, qPCR revealed that only AON #3 treatment significantly increased the amount of full-length SMN2 compared to the control at 0.5 nM or 1 nM transfection (Fig. 6e). The ratio of full-length to exon 7-deleted SMN2 of AON #3 was significantly larger than that of the all-LNA oligonucleotide (AON #16) at 1 nM transfection (Fig. 6f). These results suggest that the LNA/DNA mixmer could have better efficacies than all-LNA oligonucleotides with an equivalent sequence for SMN2 exon inclusion.Figure 6LNA-to-DNA replacement improves exon 7 inclusion activity. (a) AON sequences used for the experiment. AON #16 consists of 100% LNA. DNA base: G, A, T, C. LNA base (red): +G, +A, +T, +C. Phosphorothioated DNA base: G*, A*, T*, C*. Phosphorothioated LNA base (red): +G*, +A*, +T*, +C*. (b) RT-PCR of SMN2 and GAPDH in human SMA patient fibroblasts (GM03813). AONs were transfected at 0.5 or 1 nM concentration. Top band: exon 7-included. Bottom band: exon 7-excluded. (c) Quantification of SMN2 exon 7 inclusion in AON-treated SMA fibroblasts by RT-PCR. AONs were transfected at 0.5 nM concentration. *p < 0.05. (d) Quantification of SMN2 exon 7 inclusion in AON-treated SMA fibroblasts by RT-PCR. AONs were transfected at 1 nM concentration. ***p < 0.005. (e) Relative expression of full-length SMN2 to GAPDH measured by qPCR. The data was normalized to non-treated cells. *p < 0.05. (f) Relative expression of full-length SMN2 to ∆7 SMN2 measured by qPCR. The data were normalized to non-treated cells. ***p < 0.005. #$&Significant difference with AON #2, #3, and #16 (1 nM treatment, p < 0.005). Bars represent mean ± S.D. of three independent experiments. One-way ANOVA with a Tukey’s multiple comparison test. n.s.: not significant. M: mock control AON, NT: non-treated, H: healthy cells, B: blank.
|
study
| 100.0 |
LNA-to-DNA replacement improves exon 7 inclusion activity. (a) AON sequences used for the experiment. AON #16 consists of 100% LNA. DNA base: G, A, T, C. LNA base (red): +G, +A, +T, +C. Phosphorothioated DNA base: G*, A*, T*, C*. Phosphorothioated LNA base (red): +G*, +A*, +T*, +C*. (b) RT-PCR of SMN2 and GAPDH in human SMA patient fibroblasts (GM03813). AONs were transfected at 0.5 or 1 nM concentration. Top band: exon 7-included. Bottom band: exon 7-excluded. (c) Quantification of SMN2 exon 7 inclusion in AON-treated SMA fibroblasts by RT-PCR. AONs were transfected at 0.5 nM concentration. *p < 0.05. (d) Quantification of SMN2 exon 7 inclusion in AON-treated SMA fibroblasts by RT-PCR. AONs were transfected at 1 nM concentration. ***p < 0.005. (e) Relative expression of full-length SMN2 to GAPDH measured by qPCR. The data was normalized to non-treated cells. *p < 0.05. (f) Relative expression of full-length SMN2 to ∆7 SMN2 measured by qPCR. The data were normalized to non-treated cells. ***p < 0.005. #$&Significant difference with AON #2, #3, and #16 (1 nM treatment, p < 0.005). Bars represent mean ± S.D. of three independent experiments. One-way ANOVA with a Tukey’s multiple comparison test. n.s.: not significant. M: mock control AON, NT: non-treated, H: healthy cells, B: blank.
|
study
| 100.0 |
In this study, we designed a series of LNA-based splice-switching oligonucleotides (SSOs) targeting human SMN2 ISS-N1 in intron 7, and examined their ability to induce exon inclusion. We demonstrated that five LNA/DNA mixmers we designed efficiently induce exon inclusion and recover the expression of SMN protein in type I SMA patient fibroblasts (Figs 3 and 4). A single mismatch significantly decreased the ability to induce exon inclusion, suggesting that these LNA/DNA mixmers are highly specific to the targeted sequence (Fig. 5). This is the first report showing that LNA/DNA mixmers induce efficient exon inclusion. It might, therefore, be an attractive therapeutic strategy to treat SMA.
|
study
| 99.94 |
The results of the RT-PCRs and the qPCR indicate that transfection with the mixmers at 5 nM induces nearly 100% exon 7 inclusion in SMA fibroblast cells (Figs 2 and 3), whereas 2′MOE oligos (nusinersen) were reported to induce the exon inclusion at 100 nM in vitro 28. The effective concentration for the mixmer transfection is also lower than that of the PMO transfection (100 nM)27. Additionally, the results indicate that the 13-mer AONs are sufficient to target ISS-N1 and induce the exon inclusion whereas the 8-mer AONs are not, corresponding to the previously published data with 2′OMeP AONs18, 29. The ISS-N1 has two hnRNP A1 binding sites7, 29. The 8-mer AONs block only one of these sites, while the 13-mer AONs cover the one and more than half of the other sites. These indicate that both hnRNP A1 binding sites should be targeted by AONs to induce exon inclusion.
|
study
| 100.0 |
LNA, or 2′-O,4′-C-methylene-bridged nucleic acid (2′,4′-BNA), is an artificial nucleic acid developed independently by Wengel’s group and Obika’s group in the late 1990s (Fig. 1a)20, 21, 30. Although LNAs have been used for various gene silencing techniques, e.g. antisense gapmer, short interfering RNA, blocking of microRNA, and triplex-forming oligonucleotides22, 24, 31, 32, it has not been used as an SSO until recently. Singh et al. showed a 14-mer all-LNA oligonucleotide targeting ISS-N1 induced exon inclusion of SMN2 33. In addition, it has been reported that alternating LNA/deoxyribose oligonucleotides, or LNA/DNA mixmers, efficiently induce exon skipping in vitro 23, 34. In contrast to Singh et al.33, SSOs only containing LNAs showed very low ability to induce exon skipping in the DMD exon 5823. Shimo et al. and Yamamoto et al. hypothesized that it is probably because LNA-only-SSOs possess extremely high binding affinity to mRNAs and that the high binding affinities of LNAs reduce the dissociation rate from targeted mRNAs and prevent the efficient turnover of SSOs23, 35. Meanwhile, our data indicated that the proportion and the positions of LNA and DNA in mixmers affected the exon inclusion efficiency. Interestingly, AON #3, which is composed of LNAs with DNA being substituted for LNA at every third nucleotide position, was more effective than AON #2 (DNA substitution at every other nucleotide) and AON #16 (all-LNA oligonucleotide) (Fig. 6). It suggests that the ratio and the position of LNAs and DNAs are critical factors to determine the efficacies of LNA/DNA mixmers.
|
study
| 99.94 |
AONs containing LNAs cause hepatotoxicity in some cases because the strong binding affinity to mRNAs induces off-target effects to pre-mRNAs, particularly in the liver36. This toxicity is reported to be a sequence-specific issue rather than an issue associated with the LNA chemistry36. According to NCBI BLAST, there is no human mRNA which includes entire complementary sequences of AON #1, #2 and #3. However, AON #4 and #5 (13 mer) are identical to part of complementary sequences of dystrophin-related protein 2 (DRP2) and peroxisome proliferator activated receptor alpha (PPARA) mRNAs. Considering this and the result of Fig. 6, AON #3 might be the most suitable AONs for SMN2 exon inclusion among the mixmers we tested. The potential toxicity of each mixmer should be carefully evaluated in vivo before proceeding to clinical trials.
|
study
| 100.0 |
Our study has shown that the LNA/DNA mixmers we developed have very high efficacy in vitro. Administration of mixmers could have a significant effect in vivo, even with lower doses. From a therapeutic point of view, administration of lower concentrations and minimizing administration frequency would be desirable because of safety concerns and the cost of the treatment. Therefore, LNA/DNA mixmers are one of the promising drug candidates suitable to develop antisense oligonucleotide therapies to treat SMA.
|
study
| 99.94 |
All LNA/DNA mixmers were synthesized by Exiqon. Each mixmer sequence was examined by BLAST software to check for any potential negative off-target effects. The mock AON sequence is +T*A* +A*C* +A*C* +G*T* +C*T* +A*T* +A*C* +G*C* +C*C* +A. Phosphorothioated DNA base: G*, A*, T*, C*. Phosphorothioated LNA base: +G*, +A*, +T*, +C*.
|
study
| 99.7 |
The SMA patient fibroblasts (GM03813, Coriell NIGMS human genetic cell repository) were obtained from a male patient diagnosed with type I SMA. The cells have a homozygous deletion of exons 7 and 8 in SMN1 and have two copies of the SMN2 gene. Healthy fibroblasts (GM23815, Coriell NIGMS human genetic cell repository) were used as a control. AON transfection was performed with Lipofectamine RNAiMAX (Invitrogen) in OptiMEM I serum-reduced media (Gibco) for 48 hours.
|
clinical case
| 98.0 |
Total RNA was extracted using TRIzol Reagent (Invitrogen). RT-PCR was performed using SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen). The primers were designed to detect only SMN2 sequences (not to detect SMN1) in the SMA cell line, as the fibroblast cells from this patient have a deletion mutation of exons 7 and 8 in SMN1. The SMN2 primer sequences were 5′-CTGCCTCCATTTCCTTCTG-3′ (Forward) in exon 6 and 5′-TGGTGTCATTTAGTGCTGCTC-3′ (Reverse) in exon 8. The GAPDH primer sequences were 5′-TCCCTGAGCTGAACGGGAAG-3′ (Forward) and 5′-GGAGGAGTTTGGTCGCTGT-3′ (Reverse). The cDNA was synthesized for 5 min at 50 °C. The SMN2 exon 6–8 was amplified with 30 PCR cycles (94 °C for 15 sec, 60 °C for 30 sec, 68 °C for 25 sec). The GAPDH was amplified with 20 PCR cycles (94 °C for 15 sec, 60 °C for 30 sec, 68 °C for 20 sec).
|
clinical case
| 99.7 |
Total RNA was extracted using TRIzol Reagent (Invitrogen), and cDNA was generated using SuperScript III Reverse Transcriptase (ThermoFisher). The qPCR reaction was performed by SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and QuantStudio3 real-time PCR system (Applied Biosystems). The full-length SMN2 transcripts were amplified using the primer set: (forward, 5′-GCTATCATACTGGCTATTATATGGGTTTT-3′; reverse, 5′-CTCTATGCCAGCATTTCTCCTTAAT-3′). The ∆7 SMN2 transcripts were amplified using the primer set: (forward, 5′-TCTGGACCACCAATAATTCCCC-3′; reverse, 5′-ATGCCAGCATTTCCATATAATAGCC-3′). The expression of GAPDH was measured using the primer set (forward, 5′-GCAAATTCCATGGCACCGT-3′; reverse, 5′-AGGGATCTCGCTCCTGGAA-3′). The relative expression of full-length SMN2 to GAPDH or ∆7 SMN2 was calculated, and normalized to the non-treated cells with the ∆∆Ct algorithm.
|
study
| 99.94 |
Whole cell protein was collected using Pierce RIPA lysis buffer (Thermo Scientific) with 1x Roche cOmplete protease inhibitor. Western blotting was performed, as described37. Five μg of total protein was run per well in NuPAGE Novex 4–12% Bis-Tris Midi Protein Gels (Life Technologies). The mouse purified anti-SMN antibody (BD Biosciences) and the rabbit anti-Cofilin antibody (D3F9, Cell Signaling) were used as primary antibodies (Both were diluted to 1: 10,000, incubated for 1 hour at room temperature). HRP-conjugated goat anti-mouse IgG (H + L) (Bio-Rad) and HRP-conjugated goat anti-rabbit IgG (H + L) (Bio-Rad) were used as secondary antibodies (Both were diluted to 1:10,000). The bands were detected using Amersham ECL Select Western blotting detection kit (GE Healthcare).
|
study
| 99.94 |
Image J software (NIH) was used to quantify the band intensity. For RT-PCR of SMN2, the intensity of full-length bands and ∆7 bands were measured. The rate of exon 7 inclusion was calculated by the intensity value of (full-length)/(full-length + ∆7 SMN2). The RT-PCR, the qPCR, and the Western blotting data were analyzed by one-way repeated measures ANOVA with Tukey’s or Dunnett’s multiple comparison post hoc analysis by GraphPad Prism 7 (GraphPad software).
|
study
| 99.94 |
The ocean has been influencing the human race since ancient times. It provides food and sustenance items and plays vital role in weather system, economy and defence of the nations. More than half of the world population live within 150 km from coastline. The conventional exploring mechanism of the oceans involves sending/deploying a sampler in monitoring area of interest especially in the bottom of ocean which collects the required material and then return to surface. There is lack of real time monitoring and retrieving mechanism in case of failure or misconfiguration. Hence these limitations empower Underwater Wireless Sensor Network (UWSN) to become an appropriate candidate for the longstanding and reliable monitoring and data gathering applications. However, UWSN faces lots of challenges due to; the dynamic underwater environment, changing network topology due to water current, and shipping and aquatic life activities. It incurs the substantial cost of deployment, maintenance, and device recovery to cope with unpredictable underwater circumstances. Water absorbs lots of RF (Radio Frequency) energy hence radio communication is not feasible in underwater environments. Hence the communication in underwater is accomplished using acoustic links. Acoustic communication is mainly in low frequency typically less than 30 KHz which limits the available bandwidth and hence lower data rates are observed in underwater communications. The instruments used in underwater devices are also vulnerable to tedious subaquatic challenges, for example, algae and salt accumulation on camera lens, decreasing the effectiveness of sensors and other important devices.
|
other
| 99.9 |
Generally, large number of energy constrained nodes are deployed on different depths to sense the required parameter(s) in underwater environment. These nodes are not aware of their exact position; however, their depth can be found using depth (pressure) sensors. In underwater, nodes communicate with each other through acoustic links. As described earlier, there are some immanent characteristics of acoustic communication like, limited bandwidth (<100 KHz), larger delays, and time varying multi path fading which may cause larger doppler shift. All the deployed nodes also have energy constraint so their efficient energy consumption is of prime importance. It is almost impossible to replace or recharge the batteries of deployed nodes.
|
other
| 99.9 |
The packets generated by each node are transmitted to surface sink using broadcasting and multi-hop communication . In this communication, sending node includes its depth and the routing information in data packet and broadcasts it to all the nodes within its transmission range. However only shallower nodes (i.e., a node with depth lesser than the transmitting node) are allowed to respond to this broadcast. Only those shallower nodes which are farther than the depth threshold become eligible for forwarding. All the eligible nodes holds the packet for their respective holding time which is inversely related to forwarding function. If these packet holding nodes receive the copy of the same packet with-in their holding time they discard the packet and assume that a more eligible node has forwarded the packet. However if holding time is elapsed and no copy is received, node considers itself as the best forwarder and broadcasts that packet. This process is repeated by all nodes till the packet is reached to surface sinks. In this way each time a node with maximum value of forwarding function, gets minimum value of holding time and becomes next forwarder. This communication process involves certain terms which are needed to be explained.
|
other
| 99.9 |
Depth Threshold: Depth threshold is a global depth parameter to control the number of nodes involved in forwarding process. Greater the values of depth threshold lesser the number of nodes involved in forwarding process. Value of depth threshold is always less than the transmission range.
|
other
| 99.9 |
Forwarding Functions: Forwarding function is a routing metric which can be dependent on different parameters of nodes, like absolute depth, depth difference, residual energy, transmission range and depth threshold. Different forwarding functions have been defined in literature and included in details in the next sections.
|
other
| 99.9 |
Underwater environments are very diverse in nature. A very wide range of living and non living activities take place in the same oceanic area. Generally, this diversity increases as we move deeper into the ocean. There can be many diverse applications in which severity of the sensed value of different nodes may not be the same. The data generated by some nodes may be of more importance than others. This sternness can be of many types like, in terms of delay criticality or in terms of size of data generated. Hence the costly deployment of UWSN can not be dedicated for sensing only a single type of occurring events. These diverse applications require the routing for each type of sensed event. So it is necessary to provide an underwater network solution which considers the diverse events occurrence and appropriate response to that diverse characteristic (different delay criticality associated with different occurring events).
|
other
| 99.9 |
We have considered a diverse environment in which multiple types of events are occurring in the sensing field. These events are named as critical, very critical and normal. Critical and very critical events are delay sensitive in nature so forwarding to surface sink is planned for these types of event using respective forwarding functions in different depth regions. Normal events are sent to the mobile sinks (AUV: Autonomous Underwater Vehicle or CN: Courier Node) patrolling in the network.
|
other
| 99.9 |
Forwarding functions defined in literature use different parameters in different depth regions. However the values of different forwarding functions must be comparable with each other to ensure the steady flow of packets. Analysis on the range of previously defined forwarding function reveals the incomparable ranges of different forwarding functions. Hence the forwarding functions are redefined to ensure their comparable values in different depth region for the steady flow of packets.
|
other
| 99.8 |
Mobile sinks are deployed to collect the data from the nodes which has sensed normal events. Hence mobility of these nodes is influenced by the distribution and rate of data generation of normal nodes. We have proposed two different mobility schemes for the mobile sinks.
|
other
| 99.94 |
The rest of the paper is organized as follows: In Section 2 we have provided an extensive literature review about the protocols especially available for underwater sensor networks. The underwater channel model is explained in Section 3. Section 4 consists of problem statement and motivation in which we have identified a problem in previously defined forwarding functions by analyzing their ranges. We also present motivation for event segregation in subsection of this section. Event segregation approach, network initialization and node deployment, improvements in terms of forwarding function and holding time and mobility of courier nodes are explained in the subsections of Section 5. Performance evaluation and result discussion are explained in Section 6. Finally conclusion and future work is presented in Section 7.
|
other
| 99.75 |
As mentioned earlier, underwater network is quite different from the terrestrial networks. The routing protocols for terrestrial networks are broadly divided into two categories: proactive and reactive . In proactive protocols-like OLSR (Optimized Link State Routing) and DSDV (Dynamic Destination Sequenced Distance-Vector) -nodes periodically share their routing table information with each other, routes are established evenly irrespective of their requirement. This would cause extra overhead especially in higher lossy underwater links. Whereas in reactive protocols, like AODV (Ad-hoc On Demand Distance Vector) , its variants like MRAODV (Multiple Route AODV) and DSR (Dynamic Source Routing) ; routes are established on demand initiated from the source. In underwater networks, this route establishing mechanism would be time consuming and causes larger delay due to low propagation speed of acoustic signals. So these protocols are no longer feasible to work in harsh underwater environment . There have been many proposed protocols for routing in underwater scenarios . These protocols roughly fall into following two categories. Different aspects of these protocols are given in details.
|
other
| 97.94 |
In these protocols, position information of each node is calculated with some location finding algorithm. There have been many localization algorithms in literature . A modified version of DSR for underwater environments named as LASR (Location Aware Source Routing) was proposed in . LASR is on demand routing protocol to cope with high latency, high probability of packet loss. It takes into account location and link quality metric during routing process.
|
other
| 99.9 |
Vector Based Forwarding (VBF) was proposed in , which aims to increase network lifetime taking into account mobility of nodes. A hypothetical vector, which makes a virtual pipe of radius R from source to destination is assumed. Nodes within the range of vector are selected as candidates for forwarding process. EVBF (Efficient-VBF) is an extension and improvement in VBF, in which “desirable-ness” factor α and adaptation time Tadaptation are utilized in forwarding and selecting most suitable forwarder. This protocol is workable for both sink based queries and source generated queries. Another variant of this protocol is LE-VBF (Lifetime-Extended VBF) in which desirableness is redefined employing position and energy of the deployed node. Routing pipe radius threshold influences on the efficiency of this protocol. VBF also requires localization information and extra equipment to measure Angle of Arrival (AoA) which is an overhead and causes extra energy consumption. Nodes closer to the axis of routing pipe become hot spot and their faster energy depletion causes void regions.
|
other
| 99.8 |
In order to overcome the void area problems faced in VBF, Vector Based Void Avoidance (VBVA) is proposed in . VBVA has capability to detect a void and bypass it on demand, hence it does not need to keep record of topology information. Void avoidance is realized using two mechanisms: vector-shift and back pressure. Void is detected if a node finds no neighbour with “advance” greater than its own “advance”. Vector shift method allows nodes out of routing pipe to forward the packet and hence a shifted vector is formed. If a node cannot find neighbours to shift; it broadcasts a back pressure packet to reroute the packet with better path which has no void. However void detection process is only capable to detect it on the edge of the network and has no solution if void occurs in middle of networks due to unexpected deaths of intermediate nodes. Moreover backward flooding would cause extra over head especially in sparse networks. The performance of these protocols can be improved by identifying critical (i.e., cut-vertices) and non-critical nodes in the network using any of the algorithm such as .
|
other
| 99.1 |
Link quality based control and directional flooding protocol is proposed in named as DFR (Directional Flooding based Routing protocol). Link quality is measured in terms of Expected Transmission Count (ETX) and flooding rather than established paths is employed during routing process. Flooding zone is locally formed based on link quality of neighbour nodes. If link quality is poor it involves more nodes in forwarding process otherwise few sufficient nodes are employed. It involves two type of angles: current angle of node i; CAi and reference angle of previous sender k; RAk, in forwarding process. It also provide the mechanism for adaptive change and improvement in RAk by each forwarding node. Geographically controlled flooding zone and directional (toward sink) forwarding based on link quality ensures better packet delivery. However, this protocol requires localization information which is itself a challenge in UWSN. Acquiring link quality information for each node increases control packet broadcasting and causes extra energy consumption. DFR does not have efficient mechanism for redundant packet avoidance.
|
other
| 99.9 |
In Depth Adaptive Routing Protocol (DARP) authors considered the fact that speed of sound varies with depth and proposed shortest delay path for deep sea (about 8000 m) underwater environments. Relative Distance Based Forwarding (RDBF) is proposed in . In RDBF, fitness factor is normalized by transmission range to get the value in range of [0, 1]. Holding time is also dependent on transmission range and speed of sound in acoustic environment. Multi Path Routing (MPT) proposed in ensures balance between energy consumption and end to end delay for time critical underwater applications. Its a cross layer approach which employs: (1) power control on physical layer according to channel status and network conditions; (2) packet combining mechanism on sink for multiple copies of corrupted packets received from different paths. MPT perform well in dense deployment scenarios while suffers void regions in sparse networks.
|
other
| 99.8 |
All the above mentioned localization based protocol requires some collaborative mechanism for to find the location of the deployed nodes. Localization process in harsh underwater environment is itself a research challenge. It also requires some certain number of node to act as Anchor Nodes (AN). Anchor nodes are attached with some support and their fixed location is known. Unknown location nodes communicate with more than one anchor nodes and try to estimate their own location. Error in localization can leads to erroneous communication. Hence its preferred to have localization free routing protocol for UWSN.
|
other
| 99.9 |
This category consists of protocols which are either completely free of any localization information (based on hop count) or they may require partial localization information DBR (Depth Based Routing) requires only depth). In DBR , a receiving node from the set of potential forwarders utilizes its depth and depth threshold (a global parameter) to make the forwarding decision. Forwarding decision is done individually by each receiving node on the basis of receiving time of a certain packet and holding time of that node. Holding time is assigned to each node in such a way that node with lesser depth has less holding time and more probability to be forwarder. Other nodes are prohibited to forward the packet by assigning holding time values more than the optimal forwarder value. DBR performs well to enhance the stability period but when nodes start dying there is no mechanism to rescue and prolong the instability period (time period between the death of first and last node). Throughput of DBR in sparse networks is relatively low, which may require to implement multiple sinks. Additionally, if two nodes lies at same depth then there is no mechanism to suppress the transmission of redundant packets.
|
other
| 99.8 |
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